Chemiluminescent acridinium salts

ABSTRACT

Acridinium sulfonylamides and isomers, such as phenanthridinium sulfonylamides, may be employed in applications including chemiluminmescent immunoassays. Methods for synthesis of these compounds include contacting an amine with a sulfonylhalide to form a sulfonamide and acylating with an activated carboxylic acid of an acridine or isomer thereof. The N-sulfonyl-9-acridinium carboxamide and isomers may be conjugated to antigens, haptens, antibodies, and nucleic acids for use in chemiluminescent assays.

This application is a continuation of application Ser. No. 07/371,763,filed Jun. 23, 1989, now abandoned, which is a continuation of Ser. No.921,979, 10/22/86, abandoned.

BACKGROUND

The present invention relates in general to chemiluminescent methods andmaterials and in particular to methods and materials involvingchemiluminescent acridinium and phenanthridinium salts.

Chemiluminescence may be defined as the generation of light from achemical reaction. The mechanism of most chemiluminescent reactions isnot known in detail, but a generalized mechanism [Schuster et al.,Advances in Physical Organic Chemistry, 187-238 (1984)] may be outlined:

    A→B* →B+hν

Compound A undergoes a chemical reaction (usually oxidation) to yield aproduct in an electronically excited State ("B*"). As it returns to theground state ("B"), this product gives up energy in the form of light("hν").

Although competing dark reactions may decrease the efficiency of theoverall reaction to less than 1%, some bioluminescent systems mayachieve 60-70% efficiency, and, in many cases, limits of detection inthe femtomole (10⁻¹⁵ mole) to attomole (10⁻¹⁸ mole) range have beenrecorded.

Chemiluminescence has been used for a variety of purposes in analyticalchemistry where other methods fail to have adequate sensitivity. Inimmunodiagnostics, chemiluminescent immunoassays ("CLIA") may thus matchor exceed the sensitivity of radioimmunoassays ("RIA") or enzymeimmunoassays ("EIA") [Kricka et al., Diagnostic Medicine, 1, 45-52(1984)].

Luminol and isoluminol derivatives are the most widely usedchemiluminescent reagents for immunoassays. The light-yielding reactionis initiated by oxidation with alkaline hydrogen peroxide in thepresence of catalysts such as microperoxidase or transition metal ions.Light emission occurs at about 465 nm, which corresponds to thefluorescence emission of the product, aminopthalic acid. Aminobutylethylisoluminol ("ABEI") may be used as a label in immunoassays and iscommercially available.

A second group of chemiluminescent reagents, aryl oxalates [Gill,Aldrichimica Acta, 16, 59-61 (1983) and Catherall et al., J. Chem. Soc.Faraday Trans. 2, 80, 823-834 (1984)], have been used as commercial coldlight sources [see e.g., Tseng et al., U.S. Pat. No. 4,338,213] and inhigh performance liquid chromatography ("HPLC") detectors [Kobayashi etal., Anal. Chem., 52, 424-427 (1980) and Miyaguchi et al., J.Chromatogr., 303, 173-176 (1984)]. It is thought that these derivativesreact with hydrogen peroxide in buffered or unbuffered solvents to givea dioxetan-dione which decomposes quickly to give CO₂ in an excitedstate. Energy is then transferred by electron transfer to a fluorescermolecule which emits light.

A third group of reagents, 10-methyl-acridinium-9-carboxylic acid arylesters, are chemiluminescent in the presence of alkaline hydrogenperoxide and in the absence of a catalyst. The mechanism is thought toinvolve initial attack by a hydroperoxide anion, followed byintramolecular displacement of the phenolate (the "leaving group") togive a strained dioxetan-one. The strained dioxetan-one decomposes toCO₂ and excited N-methyl-acridone, which emits light at 430 nm.Carboxy-substituted acridinium salts have been used as labels inimmunoassays [Weeks et al., Clin. Chem., 29, 1474-79 (1983); Campell etal., European Patent Application No. 82,636; and McCapra et al., UKPatent No. GB 1,461,877]. Also, 5-methyl-phenanthridinium-6-carboxylicacid aryl esters, which are isomeric with the acridinium aryl esters,have been used as labels in immunoassays [Lin et al, European PatentApplication No. 170,415].

Despite their usefulness in immunoassays, antibody-conjugated phenyl10-methyl-9-acridiniumcarboxalates, in our hands, are unstable due tohydrolysis above pH 4.0 (-20° C. to 40° C.), losing greater than 10% oftheir activity within three days. Although acridinium esters are stablebelow pH 4.0, conjugate antibodies are often not stable in this pHrange.

In Tseng et al., supra, bis-N-alkyl-N-trifluoromethyl sulfonyloxalamides are indicated to be more stable than the corresponding arylesters and are also indicated to be as efficient. The nucleofugacity ofthe phenol and the trifluoromethyl sulfonamide are indicated to becomparable, i.e. it is indicated that each has a pK_(a) of about 7.Gill, supra, "looks forward" to the development of a particular sulfonyloxalamide as an example of an oxalate with "higher" quantum efficiency.

BRIEF DESCRIPTION OF THE DRAWINGS

The FIGURE illustrates the synthesis of a10-alkyl-N-sulfonyl-9-acridinium carboxamide according to the presentinvention.

SUMMARY OF THE INVENTION

The present invention provides chemiluminescent compounds identified bythe formula ##STR1## and isomers thereof including isomers identified bythe formula ##STR2## wherein R, R', R", X¹, X², and X³ are substituentswhich do not interfere with effective chemiluminescence, with theproviso that R-X³, R'-X² and R"-X¹ may be independently hydrogen. Morespecifically, R, R' and R" may be spacer arms and X¹, X² and X³ may beindependently members of the group consisting of hydrogen, carboxy,carboalkoxyl, carboxamido, carboaryloxy, cyano, carboximido, isocyanato,isothiocyanato, sulfo, sulfonyl halide, carbonyl halide,N-succinimidyloxycarbonyl and N-maleimide groups. Y is an appropriatecounterion and may be selected from the group consisting of sulfate,alkylsulfate, halosulfate, haloborate, haloacetate, halophosphate,phosphate and halide.

R, R', and R" may independently include a member selected from the groupconsisting of alkylene, arylene, substituted alkylene and substitutedaryl groups, such that one or more hydrogens or open valences of saidmember is replaced by an alkyl, arylene, alkenylene, alkylene,substituted alkyl, substituted alkylene, substituted aryl, alkoxy,aryloxy, halo, nitro, amino, protected amino, substituted amino,hydroxy, protected hydroxy, oxo, thio, imino, mercapto or substitutedmercapto group; or such that one or more carbon atoms of the member isreplaced by a heteroatom. The heteroatom may be selected from the groupconsisting of nitrogen, phosphorus, sulfur and oxygen.

R, R', and R" independently may also be spacer arms of the formula

    --(CH.sub.2).sub.n --

where n=0-50. Specifically, R" may be --CH₂ -- and X¹ may be --H.

The currently most preferred compounds according to the presentinvention for use in chemiluminescent immunoassays are10-methyl-N-[2-carboxyethyl]-N-tosyl-9-acridinium carboxamide,10-(3-sulfopropyl)-N-(2-carboxyethyl)-N-tosyl-9-acridinium carboxamideand 10-(3-sulfopropyl)-N-(3-sulfopropyl)-N-tosyl-9-acridiniumcarboxamide.

A method, according to the present invention, for preparation of achemiluminescent compound includes the steps of contacting an amineidentified by the formula

    X.sup.3 --R--NH.sub.2

with a sulfonylhalide identified by the formula

    W--SO.sub.2 --R'--X.sup.2

in an inert solvent in the presence of base to form a sulfonamide anionand metal ion identified by the formulas

    -M+

    X.sup.3 --R--N--SO.sub.2 --R'--X.sup.2

and acylating with an activated 9-acridinecarboxylate compound accordingto the present invention, wherein W is selected from the groupconsisting of chloro and fluoro groups, wherein M is selected from thegroup consisting of Li, Na and K, wherein the activating group isselected from the group consisting of halo, imidazolo,N-hydroxysuccinimidyl and azido groups and wherein all other symbols areas defined above.

A conjugate according to the present invention may be formed bycovalently coupling an antibody, a hapten, an antigen or apolynucleotide (e.g., DNA or RNA) to a chemiluminescent compoundaccording to the present invention, and a method for performing achemiluminescent assay comprises the step of exposing a sample to betested to the conjugate in order to detect the presence of a substancespecifically reactive with the conjugate, e.g., a specific antigen, aspecific antibody or a complementary polynucleotide (i.e., apolynucleotide which forms sequence-specific hydrogen bonds with thepolynucleotide conjugate according to the present invention).

DETAILED DESCRIPTION

The problem of acridinium aryl ester instability is approached in thepresent invention by changing the leaving group from a phenolate to asulfonamide anion. While both leaving groups have a pK_(a) of about 10,the acridinium sulfonylamide has the additional stabilization associatedwith amide bonds. This is reflected in a comparison in the infrared ofthe carbonyl stretching frequency of the aryl ester (1730 cm⁻¹) withthat of the sulfonylamide (1680 cm⁻).

A class of acridinium salts, 10-alkyl N-alkyl (aryl)sulfonyl-N-alkyl(aryl) 9-acridinium carboxamide salts, was preparedaccording to the general scheme illustrated in the FIGURE. In theFIGURE, R, R' and R" are substitutents which may function as spacerarms, solubility modifiers and/or reactivity modifiers but which do notinterfere with the chemiluminescent reaction. ("Interfere" is definedherein to mean "prevent the production of effective chemiluminescence",i.e., prevent production of chemiluminescence to the extent that thecompound is not useful for the intended application ) Also in theFIGURE, are X¹, X², X³ are substituents which may function as solubilityenhancers and/or as reactive groups for linkage to an analyte or asgroups which may be readily converted to such reactive or linker groupsby means well known to those skilled in the art. Y is a counterion inthe FIGURE.

Salts produced according to the scheme of the FIGURE have generatedlight upon oxidation with alkaline hydrogen peroxide. The compounds weremade from readily available amines (X³ --RNH₂) and sulfonyl chlorides(X² --R'SO₂ Cl). When acylated with 9-chlorocarbonyl acridine, theintermediate sulfonamide (X³ --RNH--SO₂ R'--X₂) gave a new class ofacridine compounds, which on alkylation gave the acridinium salts.Similarly, substitution of a 6-chlorocarbonyl phenanthridine for theacridine in this scheme gives rise to a new class of phenanthridiniumsalts. These acridinium and phenanthridinium salts are useful forchemiluminescent labeling of proteins, nucleic acids and small moleculesused in diagnostic testing.

Several acridinium sulfonylamides were prepared which have specificactivity and stability suitable for use in diagnostic testing,particularly in CLIA. The synthesis of these compounds allows for theintroduction of a variety of functional groups (X¹, X², X³) which may beused in antibody labeling. In addition, the kinetics of thechemiluminescent reaction may be controlled by the choice of thesubstitutents (R, R') on the sulfonamide leaving group.

The compounds were evaluated for their efficiency by diluting 20 μl of a10⁻⁹ M solution of the compound with 300 μl of 0.1N HCL, then adding 150μl of 0.03% H₂ O₂ in 0.2N NaOH to trigger the chemiluminescence.Chemiluminescence was measured on a photon-counting luminometer. Thelight output was recorded as total photon counts, from which theefficiency of each compound was calculated as counts/mole. These arerelative numbers, since the efficiency of the photon counting wasinstrument-dependent. Direct comparisons of compounds were carried outon the same instrument. The results are presented in Table 1 in whichthe structures may be identified by the formula ##STR3## wherein R"-X¹is CH₃, and R'-X² and R-X³ are as indicated in Table 1, chemiluminescentoutput is abbreviated "CTS/MOLE," the time required for total lightoutput is abbreviated "INT. TIME" and the time required to reach peaklight output is abbreviated "PEAK CTS."

                                      TABLE 1                                     __________________________________________________________________________                      CTS/MOLE                                                    R'--X.sup.2                                                                             R--X.sup.3                                                                            (X 10.sup.-18)                                                                       INT. TIME                                                                            PEAK CTS (SEC)                                __________________________________________________________________________    CF.sub.3  C.sub.6 H.sub.5                                                                       12     1      0.22                                          o-NO.sub.2 C.sub.6 H.sub.4                                                              C.sub.6 H.sub.5                                                                       10     2      0.23                                          p-Br--C.sub.6 H.sub.4                                                                   C.sub.6 H.sub.5                                                                       9      2      0.24                                          CF.sub.3  i-C.sub.3 H.sub.7                                                                     15     2      0.25                                          p-NO.sub.2 --C.sub.6 H.sub.4                                                            n-C.sub.4 H.sub.9                                                                     8      2      0.25                                          o-NO.sub.2 --C.sub.6 H.sub.4                                                            i-C.sub.3 H.sub.7                                                                     11     2      0.25                                          p-CH.sub.3 C.sub.6 H.sub.4                                                              C.sub.6 H.sub.5                                                                       9      2      0.27                                          o-NO.sub.2 C.sub.6 H.sub.4                                                              n-C.sub.4 H.sub.9                                                                     6      2      0.29                                          2,4-di-NO.sub.2 C.sub.6 H.sub.3                                                         n-C.sub.4 --H.sub.9                                                                   5      2      0.32                                          p-BrC.sub.6 H.sub.4                                                                     n-C.sub.4 H.sub.9                                                                     7      3      0.44                                          p-BrC.sub.6 H.sub.4                                                                     i-C.sub.3 H.sub.7                                                                     12     6      0.44                                          p-CH.sub.3 C.sub.6 H.sub.4                                                              n-C.sub.4 H.sub.9                                                                     5      6      0.98                                          p-CH.sub.3 C.sub.6 H.sub.4                                                              i-C.sub.3 H.sub.7                                                                     8.3    10     0.96                                          2,4,6-(C.sub.3 H.sub.7 ).sub.3 C.sub.6 H.sub.2                                          n-C.sub.4 H.sub.9                                                                     14     20     4.08                                          2,4,6-(CH.sub.3).sub.3 C.sub.6 H.sub.2                                                  n-C.sub.4 H.sub.9                                                                     5      50     11.6                                          CF.sub.3  C.sub.6 H.sub.4 CHCO.sub.2 Bn                                                         4      2      --                                            __________________________________________________________________________

All of the tested compounds were efficient (5-20×10¹⁸ counts/mole). Thespecific activity was insensitive to the nature of the R and R' groupsat locations indicated above; however, the time required to reach peaklight output and the time required for total light output differed by afactor of 50 between the fastest and slowest compounds. Electronwithdrawing groups in R and R' increased the reaction rate while bulky,electron-donating groups decreased the reaction rate. Althoughchemiluminescent compounds according to the present invention which havea chemiluminescent lifetime of 2-10 seconds are preferred forimmunoassays, compounds having shorter lifetime may be useful as asource of intense, pulsed light, and compounds having a longer lifetimemay be useful as "cold light" sources.

The stability of compounds prepared according to the present inventionwas assessed in several ways. First, the compounds were diluted tosub-nanomolar solutions in aqueous buffer at pH 5-7. The solutions wereincubated at room temperature and at 45° C., while the decrease inchemiluminescence was monitored over time. This provided qualitativeresults whereby the relative stability of the compounds was determined.Anomalous results due to non-specific adsorption of the compounds on theincubation container were minimized by the addition of detergents,protein, and the like. Unambiguous, quantitative results were obtainedby monitoring millimolar solutions of the compounds by reverse phasehigh performance liquid chromatography ("HPLC"). The stability of thesecompounds was affected by R and R' in the same way as were the kineticsof the chemiluminescence reaction, i.e. electron withdrawing groupsdestabilized and bulky electron donating groups stabilized thecompounds.

Although other techniques may be employed to label antibodies, the NHSactivation method is presently preferred. Other materials which functionwell according to the present invention include polyclonal antibodies,monoclonal antibodies, Fab antibody fragments, all of which arehereinafter included in the general term "antibody," haptens, antigens,nucleic acid probes, and non-antibody, binding-proteins capable ofbinding complementary small molecular weight analytes (for example,folate binding protein which binds folic acid, and intrinsic factor,which binds Vitamin B₁₂). Antibody conjugates retain more than 80%chemiluminescence after being heated at 45° C. for four weeks.

A solid phase sandwich immunoassay system for assaying hepatitis Bsurface antigen ("HBsAg") (Abbott Laboratories, Abbott Park, Ill.) wasemployed to compare CLIA according to the present invention with RIA.The type of antibody-coated bead, diluent, incubation conditions,washing conditions and antibody preparation were the same except thatthe antibody was labeled with ¹²⁵ I by the chloramine T method for RIAand labeled with NHS-activated N-sulfonyl-9-acridinium carboxamide forCLIA.

A solid phase sandwich immunoassay for human thyroid stimulating hormone(hTSH) was used to compare CLIA with EIA (Abbott Laboratories, AbbottPark, Ill.). The EIA employed a horseradish peroxidase ("HRPO")-labelledantibody while the CLIA used an NHS-activated N-sulfonyl-9-acridiniumcarboxamide.

The present invention is more specifically described in the followingexamples. In Example 1, the preparation of sulfonamides which are usefulin constructing compounds according to the present invention is setforth. Example 2 includes a description of the preparation ofN-sulfonyl-9-acridinecarboxamides according to the present invention. InExample 3, the preparation of 10-methyl N-sulfonyl-acridiniumcarboxamides is described. Examples 4-6 contain descriptions ofsyntheses of p-toluenesulfonyl (tosyl) compounds according to thepresent invention. In Example 7, the preparation of acridinecarboxamidesis illustrated.

Example 8-10 contain methods for synthesis of some acridiniumcarboxamides and products thereof according to the present invention. InExample 11, an evaluation of the chemiluminescence ofN-sulfonylacridinium carboxamide compounds according to the presentinvention is provided. Example 12 includes a report of a stability testof an acridinium carboxamide according to the present invention. InExample 13, the temperature and pH stability of two acridiniumcarboxamides according to the present invention is compared to thetemperature and pH stability of an acridiumcarboxylate. Example 14 is adescription of a method for conjugating an antibody, specifically animmunoglobulin G ("IgG") antibody, with a compound according to thepresent invention. The results of a heat stability study of a conjugateaccording to Example 14 are presented in Example 15. Example 16 includesa description of the preparation of anti-HBsAg acridinium-labeledconjugate as well as a comparison of the sensitivity observed in CLIAand RIA assays employing those conjugates. In Example 18, the synthesisof a phenanthridinium compound according to the present invention isdescribed. Example 17 describes an anti-hTSH acridinium-labeledconjugate along with a comparison to an EIA system.

EXAMPLE 1 General Method for Preparation Of Sulfonamides

Amine starting materials for compounds 1-13 and 17-21 are available fromAldrich Chemical Co., Milwaukee, Wis. For compounds 14-16 and 22-25, theappropriate aminocarboxylic acid (as obtained from Aldrich Chemical Co.,Milwaukee, Wis.) was esterified according to standard, publishedprocedures to provide the starting materials.

In order to prepare a sulfonamide according to the present invention,the corresponding amine (200 mole percent) was dissolved in anhydrousmethylene chloride, and was treated dropwise at 0° C. with a solution(100 mole percent) of the sulfonyl chloride or anhydride. The solutionwas poured into anhydrous ether (5 volumes), washed with 1.4M H₃ PO₄ (25ml) and then brine (25 ml), and dried over MgSO₄. After filtering andevaporating, crude sulfonamides were crystallized from an appropriatesolvent.

The following sulfonamides were prepared in this manner. In thedescription accompanying the name of each compound, the abbreviation"MS" identifies peaks, such as the base peak ("M⁺ ") in the massspectrum at a location (i.e., at an m/e) specified by the symbol "@". Amelting point ("M_(p) ") or an indication that the material is a liquidat room temperature (e.g. "oil") or decomposes before melting("decomp.") may be provided. Each compound is identified by a "compoundnumber" (1-25 in this Example) followed by an "identifying number" (e.g.13513-227) and a chemical name.

    ______________________________________                                        1.    13513-227 N-Phenyl-p-toluenesulfonamide                                                 MS M.sup.+  @ 247                                                             M.sub.p 100-102° C.                                    2.    13513-228 N-Phenyl-p-bromobenzenesulfonamide                                            MS M.sup.+  @ 311                                                             M.sub.p 115-117° C.                                    3.    13513-229 N-Phenyl-o-nitrobenzenesulfonamide                                            MS M.sup.+  @ 278                                                             M.sub.p 112-113° C.                                    4.    13513-231 N-Phenyl-p-nitrobenzenesulfonamide                                            MS M.sup.+  @ 278                                                             M.sub.p 168-170° C.                                    5.    13513-232 N-Phenyl-2,4-dinitrobenzene-                                                  sulfonamide                                                                   MS M.sup.+  @ 323                                                             M.sub.p 110-113° C.                                    6.    13513-233 N-Phenyl-trifluoromethane-                                                    sulfonamide                                                                   MS M.sup.+  @ 225                                                             M.sub.p 65-67° C.                                      7.    13514-001 N-Isopropyl-p-                                                                toluenesulfonamide                                                            MS M.sup.+  @ 213                                                             M.sub.p 50-51° C.                                      8.    13514-002 N-Isopropyl-p-                                                                bromobenzenesulfonamide                                                       MS M.sup.+  @ 277                                                             M.sub.p 95-96° C.                                      9.    13514-003 N-Isopropyl-o-                                                                nitrobenzenesulfonamide                                                       MS M.sup.+  @ 244                                                             M.sub.p 119-120° C.                                    10.   13514-004 N-Isopropyl-                                                                  trifluoromethanesulfonamide                                                   MS (M - 1) @ 190                                                              oil                                                           11.   13514-006 N-Isopropyl-p-                                                                nitrobenzenesulfonamide                                                       MS M.sup.+  @ 244                                                             M.sub.p 113-114° C.                                    12.   13514-025 N-Butyl-2,4,6-                                                                trimethylbenzenesulfonamide                                                   MS M.sup.+  @ 255                                                             M.sub.p 45° C.                                         13.   13514-026 N-Butyl-2,4,6,-                                                               trisopropylbenzenesulfonamide                                                 MS M.sup.+  @ 339                                                             M.sub.p 104° C.                                        14.   13514-032 Benzyl 6-(N-tosylamino)-                                                      hexanoate                                                                     MS M.sup.+  @ 375                                                             oil                                                           15.   13514-057 t-Butyl N-tosyl-β-alanine                                                MS M.sup.+  @ 242 (M - 57)                                                    oil                                                           16.   13514-058 Benzyl 5-(N-tosylamino)-pentanoate                                            MS M.sup.+  @ 361                                                             oil                                                           17.   13513-170 N-Butyl-p-toluenesulfonamide,                                                 MS M.sup.+  @ 227                                                             M.sub.p 42-44° C.                                      18.   13513-173 N-Butyl-p-bromobenzenesulfonamide,                                            MS M.sup.+  @ 241                                                             M.sub.p 53-54° C.                                      19.   13513-172 N-Butyl-o-nitrobenzenesulfonamide,                                            MS M.sup.+  @ 258                                                             M.sub.p 58-60° C.                                      20.   13513-174 N-Butyl-p-nitrobenzenesulfonamide                                             MS M.sup.+  @ 258                                                             M.sub.p 80-81° C.                                      21.   13513-213 N-Butyl-2,4-dinitrobenzene                                                    sulfonamide,                                                                  MS M.sup.+  @ 304                                                             M.sub.p 60-62° C.                                      22.   13513-085 Benzyl 6-(N-trifluoromethyl-                                                  sulfonylamino)-hexanoate                                                      oil                                                           23.   13513-083 Benzyl N-(trifluoromethylsulfonyl)-                                           4-(carboxymethyl) aniline                                     24.   14973-1A  Benzyl N-(5-carboxypentyl)-p-                                                 bromobenzenesulfonamide                                                       MS M.sup.+  @ 439                                                             M.sub.p 52-56° C.                                      25.   14973-37A Benzyl N-(5-carboxypentyl)-p-                                                 nitrobenzenesulfonamide                                                       MS M.sup.+  @ 406                                                             M.sub.p 86-88° C.                                      ______________________________________                                    

EXAMPLE 2 Preparation of N-sulfonyl-9-acridinecarboxamides

Freshly sublimed potassium tert-butoxide (200 mole percent) andtri-n-butylbenzylammonium bromide (1 mole percent) were suspended intoluene under nitrogen. A selected sulfonamide (200 mole percent) wasadded, the mixture was stirred for 10-30 minutes before evaporating todryness and the dried material resuspended in the solvent.[Alternatively, the phase transfer catalyst may be omitted and anappropriate anion may be generated in tetrahydrofuran.] After theaddition of 9-chlorocarbonylacridine hydrochloride (100 mole percent),the reaction mixture was stirred for 3 to 14 hours at room temperatureuntil no further change was noted by thin-layer chromatography ("TLC").The reaction solution was diluted with ethyl ether (10 volumes) andwashed with brine (25 ml). After drying over MgSO₄, filtering andevaporating, the crude product was chromatographed (on a Chromatotron™chromatograph [available from Harrison Research, Palo Alto, Calif.]using a 2 mm silica rotor and employing an ethylacetate/hexanegradient). The fractions containing the product were collected,evaporated and crystallized from ether/heptane (i.e., the fractions weredissolved in ether followed by the addition of heptane until the mixturebecame cloudy).

The following compounds were prepared from starting materials asindicated in brackets wherein starting materials prepared herein areidentified by the number associated with them in Example 1 or in thisexample, and wherein a commercial source is provided in brackets foreach identified starting material not synthesized herein. All othernotations are explained in Example 1.

    ______________________________________                                        26.   13513-234  N-Phenyl-N-p-toluenesulfonyl-                                                 9-acridinecarboxamide                                                         [compound 1]                                                                  MS M.sup.+  @ 452                                                             M.sub.p 200° C.                                       27.   13513-236  N-Phenyl-N-p-bromobenzene-                                                    sulfonyl 9-acridinecarboxamide                                                [compound 2]                                                                  MS M.sup.+  @ 516                                                             M.sub.p 218-219° C.                                   28.   13513-240  N-Phenyl-N-o-nitrobenzene-                                                    sulfonyl 9-acridinecarboxamide                                                [compound 3]                                                                  MS M.sup.+  @ 483                                                             M.sub.p 197-200° C.                                   29.   13513-242  N-Phenyl-N-p-nitrobenzene-                                                    sulfonyl-9-acridinecarboxamide                                                [compound 4]                                                                  MS M.sup.+  @ 483                                            30.   13513-243  N-Phenyl-N-trifluoromethane-                                                  sulfonyl-9-acridinecarboxamide                                                [compound 6]                                                                  MS M.sup.+  @ 430                                                             M.sub.p 162° C.                                       31.   13514-007  N-Isopropyl-N-p-toluene-                                                      sulfonyl-9-acridinecarboxamide                                                [compound 7]                                                                  MS M.sup.+  @ 418                                                             M.sub.p 163-164° C.                                   32.   13514-009  N-Isopropropyl-N-p-                                                           bromobenzenesulfonyl-9-                                                       acridinecarboxamide                                                           [compound 8]                                                                  MS M.sup.+  @ 482                                                             M.sub.p 205° C.                                       33.   13514-012  N-Isopropyl-N-o-nitrobenzene-                                                 sulfonyl-9-acridinecarboxamide                                                [compound 9]                                                                  MS M.sup.+  @ 449                                                             M.sub.p 215° C.                                       34.   13514-001  N-Isopropyl-N-trifluoromethane                                                sulfonyl-9-acridinecarboxamide                                                [compound 10]                                                                 MS M.sup.+  @ 396                                            35.   13514-028  N-Butyl-N-2,4,6,-trimethyl-                                                   benzenesulfonyl-9-acridine-                                                   carboxamide                                                                   [compound 12]                                                                 MS M.sup.+  @ 460                                                             M.sub.p 88-90° C.                                     36.   13514-031  N-Butyl-2,4,6-triisopropylbenzene-                                            sulfonyl-9-acridinecarboxamide                                                [compound 13]                                                                 MS M.sup.+  @ 544                                            37.   13514-042  Benzyl N-tosyl-N-(5-carboxypentyl)-9-                                         acridinecarboxamide                                                           [compound 14]                                                                 MS M.sup.+  @ 550                                                             oil                                                          38.   13514-062  Benzyl N-tosyl-N-(4-carboxybutyl)-9-                                          acridinecarboxamide                                                           [compound 16]                                                                 MS M.sup.+  @ 566                                            39.   13514-069  t-Butyl N-tosyl-N-(2-carboxyethyl)-                                           9-acridinecarboxamide                                                         [compound 15]                                                                 MS M.sup.+  @ 504                                                             M.sub.p  157-158° C.                                  40.   13513-186  N-Butyl-N-p-toluenesulfonyl-9-                                                acridinecarboxamide                                                           [compound 17]                                                                 MS M.sup.+  @ 432                                                             M.sub.p 122-123° C.                                   41.   13513-191  N-Butyl-N-o-nitrophenylsulfonyl-                                              9-acridinecarboxamide                                                         [compound 19]                                                                 MS M.sup.+  @ 463                                                             M.sub.p 170° C.                                       42.   13513-195  N-Butyl-N-p-nitrophenylsulfonyl-9-                                            acridinecarboxamide                                                           [compound 20]                                                                 MS M.sup.+  463                                                               M.sub.p 210° C.                                       43.   13513-218  N-Butyl-N-(2,4-dinitrophenylsulfonyl)-                                        9-acridinecarboxamide                                                         [compound 21]                                                                 MS M.sup.+  @ 508                                                             M.sub.p 95° C.                                        44.   14973-9C   Benzyl N-(5-carboxypentyl)-N-p-                                               bromobenzenesulfonyl-9-                                                       acridinecarboxamide                                                           [compound 24]                                                                 MS (M + H) @ 645                                             45.   14973-40C  Benzyl N-(5-carboxypentyl)-N-p-                                               nitrobenzenesulfonyl-9-                                                       acridinecarboxamide                                                           [compound 25]                                                                 MS (M + H) @ 645                                             46.   14973-88A  N-p-Toluenesulfonyl-9-                                                        acridinecarboxamide                                                           [p-toluene sulfonamide (Aldrich)]                                             M.sub.p 276° C.                                       47.   14973-21C  N-Allyl-N-p-toluenesulfonyl-9-                                                acridinecarboxamide                                                           [compound 46]                                                                 M.sub.p 136-138° C.                                   48.   13513-202  N-Butyl-N-p-bromobenzenesulfonyl-                                             9-acridinecarboxamide                                                         MS M.sup.+  @ 496/498                                                         M.sub.p 148-149° C.                                   ______________________________________                                    

EXAMPLE 3 Preparation of 10-Methyl N-sulfonylacridinium carboxamides

Methylation of N-sulfonylacridine carboxamides was performed accordingto the following procedure. Each acridine sulfonylamide was dissolved inanydrous methylene chloride. Anhydrous Na₂ CO₃ (5 X weight of thesulfonamide) was added followed by methyl triflate (20 X weight of thesulfonamide). The suspension was stirred under nitrogen for 14-48 hoursat room temperature to 40° C. The reaction was monitored by TLC (reversephase). The product was obtained after filtration and evaporation of thesolvent and of excess methyl triflate. Purification was achieved bytriturating the solid residue with hot benzene or by reverse phase HPLC.

The following compounds were prepared, and they are described accordingto the numerals, symbols and abbreviations which are explained inExample 1 or in Example 2.

    ______________________________________                                        49.  13513-246  10-Methyl-N-phenyl-N-p-                                                       toluenesulfonyl-9-                                                            acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 26]                                                                 MS M.sup.+  @ 467                                                             M.sub.p 210-24° C. (decomp.)                           50.  13513-247  10-Methyl-N-phenyl-N-p-                                                       bromobenzenesulfonyl-9-                                                       acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 27]                                                                 MS M.sup.+  @ 531, 533                                                        M.sub.p 240° C. (decomp.)                              51.  13513-248  10-Methyl-N-phenyl-o-nitro-                                                   benzenesulfonyl-9-                                                            acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 28]                                                                 MS M.sup.+  @ 490                                                             M.sub.p 248-50° C. (decomp.)                           52.  13513-249  10-Methyl-N-phenyl-N-                                                         trifluoromethanesufonyl-9-                                                    acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 30]                                                                 MS M.sup.+  @ 445                                             53.  13513-250  10-Methyl-N-phenyl-p-                                                         nitrobenzenesulfonyl-9-                                                       acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 29]                                                                 MS M.sup.+  @ 484                                             54.  13514-013  10-Methyl-N-isopropyl-N-p-                                                    toluenesulfonyl-9-                                                            acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 31]                                                                 MS M.sup.+  @ 433                                                             M.sub.p 214° C.                                        55.  13514-014  10-Methyl-N-isopropyl-N-p-                                                    bromobenzenesulfonyl-9-                                                       acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 32]                                                                 MS M.sup.+  @ 497/499                                                         M.sub.p 200° C. (decomp)                               56.  13514-018  10-Methyl-N-isopropyl-N-o-                                                    nitrobenzenesulfonyl-9-                                                       acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 33]                                                                 MS M.sup.+  @ 464                                             57.  13514-021  10-Methyl-N-isopropyl-N-                                                      trifluoromethanesulfonyl-9-                                                   acridinium carboxamide                                                        trifluormethanesulfonate                                                      [compound 34]                                                                 MS M.sup.+  @ 411                                             58.  13514-037  10-Methyl-N-butyl-N-(2,4,6-                                                   trimethylbenzenesulfonyl-                                                     9-acridinium carboxamide                                                      trifluoromethanesulfonate                                                     [compound 35]                                                                 MS M.sup.+  @ 475                                                             M.sub.p 227° C. (decomp.)                              59.  13514-038  10-Methyl-N-butyl-N-(2,4,6                                                    triisopropylbenzenesulfonyl-9-                                                acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 36]                                                                 MS M.sup.+  @ 559                                                             M.sub.p 231° C. (decomp.)                              60.  13514-044  Benzyl 10-methyl-N-tosyl-                                                     N-(5-carboxypentyl)-9-                                                        acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 37]                                                 61.  13514-079  t-Butyl 10-methyl-N-tosyl-                                                    N-(2-carboxyethyl)-9-                                                         acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 39]                                                                 MS M.sup.+  @ 519                                                             M.sub.p 207° C. (decomp.)                              62.  13513-211  10-Methyl-N-butyl-N-p-                                                        toluenesulfonyl-9-                                                            acridinium carboxamide                                                        trifluoromethanesulfonate.                                                    [compound 40]                                                                 MS M.sup.+  @ 447                                             63.  13513-212  10-Methyl-N-butyl-N-p-                                                        bromobenzenesulfonyl-9-                                                       acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 48]                                                                 MS M.sup.+  @ 511                                                             M.sub.p 126° C.                                        64.  13513-215  10-Methyl-N-butyl-N-o-                                                        nitrophenylsulfonyl-9-                                                        acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 41]                                                                 MS M.sup.+  @ 478                                                             M.sub.p 232-234° C.                                    65.  13513-216  10-Methyl-N-butyl-N-p-                                                        nitrophenysulfonyl-9-                                                         acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 42]                                                                 MS M.sup.+  @ 478                                                             M.sub.p 201° C.                                        66.  13513-230  10-Methyl-N-butyl-N-(2-4                                                      dinitrophenylsulfonyl)-9-                                                     acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 43]                                                                 MS M.sup.+  @ 523                                                             M.sub.p 215-220° C.                                    67.  14973-31B  10-Methyl-N-allyl-N-p-                                                        toluenesulfonyl-9-                                                            acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 47]                                                                 MS M.sup.+  2 @ 433                                           68.  14973-47A  Benzyl 10-methyl-N-(5-                                                        carboxypentyl)-N-p-                                                           nitrobenzenesulfonyl-9-                                                       acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 45]                                                                 MS M.sup.+  @ 626                                                             M.sub.p 139-141° C.                                    69.  14973-90A  10-Methyl-N-methyl-N-p-                                                       toluenesulfonyl-9-                                                            acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 46]                                                                 MS M.sup.+  @ 405                                             70.  14973-25A  Benzyl 10-methyl-N-(5-carboxypentyl)-N-                                       (o-bromobenzenesulfonyl)-9-                                                   acridinium carboxamide                                                        [compound 44]                                                 ______________________________________                                    

EXAMPLE 4 Synthesis of10-methyl-N-tosyl-N-(6-hexanoyl-N-hydroxysuccinimido)-9-acridiniumcarboxamide trifluoromethanesulfonate

Compound 37 (450 mg, 0.78 mmoles) was treated with ml of 31% HBr inacetic acid at 50° C. for 2 hours under N₂. The solution was poured into30 ml of water and cooled. Carboxylic acid compound 71, 13514-045[N-tosyl-N-(5-carboxypentyl)-9-acridinecarboxamide] was separated byfiltration.

Compound 71 (100 mg., 0.2 mmol) was dissolved in dry methylene chloride(5 ml) and treated with N-hydroxysuccinimide (23 mg, 0.2 mmol) anddicyclohexylcarbodiimide (41 mg) under N₂ for 12 hours. After reacting,the solution was filtered and then evaporated to dryness to yield anactive ester, compound 72, 13514-052[N-tosyl-N-(6-hexanoyl-N-hydroxysuccinimido)-9-acridinecarboxamide].

Compound 72 was methylated as in Example 3 to give compound 73.Compounds 71, 72, and 73 are described below using the numerals, symbolsand abbreviations which are explained in Example 1.

    ______________________________________                                        71.    13514-045  N-Tosyl-N-(5-carboxypentyl)-9-                                                acridinecarboxamide                                                           [compound 37]                                                                 MS M.sup.+ @ 240                                                              M.sub.p 150-152° C.                                  72.    13514-052  N-Tosyl-N-(6-hexanoyl-                                                        N-hydroxysuccinimido)-9-                                                      acridinecarboxamide                                                           [compound 71]                                                                 MS M.sup.+ @ 588                                            73.    13514-054  10-Methyl-N-tosyl-N-(6-                                                       hexanoyl-N-hydroxysuccinimido)-                                               9-acridinumcarboxamide                                                        trifluoromethanesulfonate                                                     [compound 72]                                               ______________________________________                                    

EXAMPLE 5 Synthesis of10-Methyl-N-tosyl-N-(5-pentanoyl-N-hydroxysuccininimido)-9-acridiniumcarboxamide trifluoromethanesulfonate

Compound 38, 13514-062, was treated as in Example 4 and yielded compound74, 13514-065 [N-tosyl-N-(4-carboxybutyl)-9-acridinecarboxamide].

Compound 74 was coupled to N-hydroxysuccinimide, as in Example 4, togive compound 75,13514-067,N-tosyl-N-(5-pentanoyl-N-hydroxysuccinimido)-9-acridinecarboxamide. Thiscompound was methylated as in Example 3 to give compound 76, 13514-078[10-methyl N-tosyl-N-(5-pentanoyl-N-hydroxysuccinimide)-9-acridiniumcarboxamide trifluoromethanesulfonate].

Compounds 74, 75 and 76 are described using the numerals, symbols andabbreviations which are explained in Example 1.

    ______________________________________                                        74.   13514-065   N-Tosyl-N-(4-carboxybutyl)-9-                                                 acridinecarboxamide                                                           MS M.sup.+ @ 476                                                              M.sub.p 152-155° C.                                  75.   13514-067   N-Tosyl-(5-pentanoyl N-hydroxy                                                succinimido)-9-acridinecarboxamide                                            [compound 74]                                                                 MS M.sup.+ @ 573                                            76.   13514-078   10-Methyl-N-tosyl-N-(5-                                                       pentanoyl-N-hydroxy-                                                          succinimido)-9-                                                               acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 75]                                               ______________________________________                                    

EXAMPLE 6 Synthesis of 10-methyl-N-tosyl-N-(2-carboxyethyl-9-acridiniumcarboxamide trifluoromethanesulfonate

Compound 61, 13514-079 (50 mg, 0,072 mmol) was dissolved in 2 ml oftrifluoroacetic acid ["TFA"] at 0° C. under N₂. After stirring for 15minutes, the TFA was evaporated and the residue was recrystallized frommethanol/ether (i.e., the residue was dissolved in methanol, addingether until cloudy). Alternatively, compound 61, was refluxed in 1N HClfor 3 hours. The aqueous solution was evaporated to dryness to leave aresidue, and the residue was purified by preparative reverse phase HPLC.Compound 77, 13514-081 [10-methylN-tosyl-N-(2-carboxyethyl)-9-acridinium carboxamide] resulted fromeither approach. Compound 77 is described using the numerals, symbolsand abbreviations which are explained in Example 1.

    ______________________________________                                        77.    13514-081  10-Methyl-N-tosyl-N-                                                          (2-carboxyethyl)-9-                                                           acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 61]                                                                 MS (M + 14) @ 477; M.sup.+ @ 463                                              M.sub.p 227° C. (decomp.)                            ______________________________________                                    

EXAMPLE 7 Preparation of Acridinecarboxamides

An amine (110 mole percent) and triethylamine (220 mole percent) weredissolved in methylene chloride. One hundred mole percent of9-chlorocarbonyl acridine was added dropwise as a solution in methylenechloride. The reaction was stirred under N₂ for 3 hours. The solutionwas filtered through silica gel and the filtrate was evaporated to leavea residue. The residue was then recrystallized from an appropriatesolvent (isopropyl ether for compound 78 and ethyl ether for compound79).

The following amides were prepared, and are described using thenumerals, symbols and abbreviations which are explained in Example 1.

    ______________________________________                                        78.    14973-15A  N-Allyl-9-acridinecarboxamide                                                 [Allyl amine (Aldrich)]                                                       MS M.sup.+ @ 262                                                              M.sub.p 192° C.                                      79.    14973-6A   Benzyl N-(5-carboxypentyl)-9-                                                 acridinecarboxamide                                                           [6-Amino caproic acid (Aldrich)]                                              MS M.sup.+ @ 458                                                              M.sub.p 86° C.                                       ______________________________________                                    

EXAMPLE 8 Synthesis of Acridinium carboxamides

An ester (either compound 44 or compound 68) was added to a 1N HClsolution and refluxed for 3-4 hours. Upon cooling, the suspension waseither filtered and the product collected, or the suspension wasextracted with a chloroform:isopropanol (3:2) mixture, which providedthe desired product (compound 80 or 81, respectively) on evaporation.Compounds 80 and 81 are described using the numerals, symbols andabbreviations which are explained in Example 1.

    ______________________________________                                        80.   14379-27A   10-Methyl-N-(5-carboxypentyl)-N-                                              p-bromobenzenesulfonyl-9-                                                     acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 44]                                                                 MS M.sup.+ @ 569, 571                                                         M.sub.p 148-150° C.                                  81.   14973-51A   10-Methyl-N-(5-carboxypentyl)-N-                                              p-nitrobenzenesulfonyl-9-                                                     acridinium carboxamide                                                        trifluoromethanesulfonate                                                     [compound 68]                                                                 MS M.sup.+ @ 536                                            ______________________________________                                    

EXAMPLE 9 Synthesis of10-(3-sulfopropyl)-N-tosyl-N-(2-carboxyethyl)-9-acridinium carboxamide

Propane sultone (260 mole percent) was heated with t-butylN-tosyl-N-(2-carboxyethyl)-9-acridinecarboxamide (compound 39,13514-069) at 110°-120° C. for 2 hours. After cooling, the solid masswas taken up in methanol and filtered. The filtrate was evaporated todryness and the residue triturated with benzene to remove un-quaternizedmaterial.

The crude product compound was treated with trifluoracetic acid at 0° C.then allowed to warm to 25° C. over a period of 15 minutes. The residueobtained upon evaporation was purified chromatographically onpreparative thick layer chromatography plates (C-18 PLKC 18F, 20×20 cm,1000M, as available from Whatman, Clifton, N.J.), eluted with 70 partsmethanol/30 parts 0.5% aqueous acetic acid, and further purified by ionexchange on Cellex-D™ resin [BioRad Laboratories, Richmond, Calif.]using 8% formic acid to elute the product, compound 82, which isdescribed below using the numerals, symbols and abbreviations which areexplained in Example 1.

    ______________________________________                                        82.    14496-243  10-(3-sulfopropyl)-N-tosyl-N-(2-                                              carboxyethyl)-9-                                                              acridinium carboxamide                                                        [compound 39]                                                                 MS M.sup.+ @ 572                                            ______________________________________                                    

EXAMPLE 10 Synthesis of10-(3-sulfopropyl)-N-tosyl-N-(3-sulfopropyl)-9-acridinium carboxamide

Fifty milligrams of N-tosyl-9-acridinecarboxamide (compound 46,14973-88A) were heated at 140°-150° C. under argon in a sealed tube with500 mg of propane sultone for 3 hours. After cooling, excess propanesultone was removed by trituration with benzene (5 ml×3). The crudeproduct was purified by anion exchange chromatography using BioRadAG-1-X4 formate form [BioRad Laboratory, Richmond, Calif.], eluted witha gradient of aqueous formic acid. The product, compound 83, isdescribed below using the numerals, symbols and abbreviations explainedin Example 1.

    ______________________________________                                        83.    30253-020  10-(3-Sulfopropyl)-N-tosyl-N-(3-                                              sulfopropyl)-9-                                                               acridinium carboxamide.                                                       [compound 46]                                                                 MS M + H @ 621.                                             ______________________________________                                    

EXAMPLE 11 Evaluation of N-sulfonylacridinium carboxamideChemiluminescence

Acridinium compounds to be tested for chemiluminescence were dissolvedin dimethyl formamide ("DMF") and then diluted with 0.05M sodium citrate(pH 5.0) or 0.05M sodium phosphate (pH 7.0) buffer to give solutions ofabout 3×10⁻⁹ M. Twenty microliters of each buffered solution was dilutedwith 300 μl of 0.1N HCl and chemiluminscence was triggered with 150 μlof 0.03% H₂ O₂ in 0.2N NaOH.

The light generated was recorded on a photon counter luminometer over a10 second interval except where a longer interval is indicated inTable 1. The specific activity of each compound is provided in the formof counts/mole in Table 1.

                  TABLE 2                                                         ______________________________________                                        Compound No.  Identifying No.                                                                             Counts/Mole                                       ______________________________________                                        49            13513-246     9.4 × 10.sup.18                             50            13513-247     9 × 10.sup.18                               51            13513-248     1 × 10.sup.19                               50            13513-249     1.2 × 10.sup.19                             53            13513-250     1 × 10.sup.19                               54            13514-013     8.3 × 10.sup.18                             55            13514-014     1.25 × 10.sup.19                            56            13514-018     1.1 × 10.sup.19                             57            13514-021     1.5 × 10.sup.19                             58            13514-037     5.2 × 10.sup.18                                                         (50 secs)                                         59            13514-038     1.4 × 10.sup.19                                                         (20 secs)                                         62            13513-211     5 × 10.sup.18                               63            13513-212     7 × 10.sup.18                               64            13513-215     6.1 × 10.sup.18                             65            13513-216     8 × 10.sup.18                               66            13513-230     5 × 10.sup.18                               ______________________________________                                    

EXAMPLE 12 Stability Test of Compound 62 (13513-211)

Compound 62 (2 mg) was dissolved in 1 ml of methanol. Fifty microlitersof this solution were added to each of the following buffers:

1) 500 microliters of 0.05M sodium phosphate, pH 5.0

2) 500 microliters of 0.05M sodium phosphate, pH 5.5

3) 500 microliters of 0.05M sodium phosphate, pH 6.0

4) 500 microliters of 0.05M sodium phosphate, pH 6.5

5) 500 microliters of 0.05M sodium phosphate, pH 7.0.

Each solution was analyzed on a Perkin-Elmer Series 4 HPLC using areverse phase column (C-18 μ Bondapak, 3.9 mm×30 cm, available fromWaters Associates, Milford, Mass.). The elution was done with 75%methanol and 25% 5 mM pentanesulfonic acid in 1% aqueous acetic acid ata flow rate of 1 ml/min. The effluent was monitored at 254 nm.

After 4 weeks at room temperature, the solutions at pH 5.0, pH 5.5 andpH 6.0 showed no sign of decomposition, while at pH 6.5 and at pH 7.0,20% and 70% decomposition were seen, respectively.

EXAMPLE 13 Comparison of Temperature And pH Stabilities of AcridiniumCompounds in Bufferat pH 7.2

Three different acridinium compounds, compound 62, 13513-211, a compoundidentified by the number 13514-020[4-(carbobenzyloxymethyl)-phenyl-10-methyl-9-acridinium carboxylatetrifluoromethanesulfonate] (compound 81) as prepared as in Weeks, etal., Clin. Chem., 29, 1474-79 (1983), and compound 83, 30253-020, werecompared for temperature and pH stability. The comparison was carriedout in methanol or water at a concentration of 1.0 mg/ml (which isapproximately equivalent to 1.6×10⁻³ M). Each of the samples was diluted1:100 in an acid solution containing one part of 0.1N HCl plus one partphosphate-buffered saline ("PBS") pH 6.8 with 0.01% Tween20®(polyoxyethylene sorbitan monolaurate available from Sigma ChemicalCompany, St. Louis, Mo.). The final pH of the diluent solution wereabout 1.5. The molarity of each of these solutions was 1.6×10⁻⁵ M.

Each of the solutions was scanned to record a UV-visible absorptionspectrum in order to determine molar extinction coefficients and inorder to detect any appreciable differences in the absorbance spectra.The UV-visible absorption spectra of these acridinium compounds have thecharacteristics presented in Table 2.

                  TABLE 3                                                         ______________________________________                                        Compound  Identifying            Observed                                     No.       No.         Wavelength Absorbance                                   ______________________________________                                        62        13513-211   263     nm   1.40                                                             369     nm   0.286                                      83        30253-020   263.5   nm   1.42                                                             370     nm   0.304                                      87        13514-020   262     nm   1.72                                                             368     nm   0.334                                      ______________________________________                                    

For all three compounds, ε₃₇₀ ≅18,000 and ε₂₆₃ ≅87,000.

These spectra indicate that there is very little difference either inUV-visible absorbance or in molar extinction coefficients among thesethree compounds. In fact, within the limitations of experimental error,few or no spectral differences were observed.

The 1.6×10⁻⁵ M stock solutions of the three compounds were seriallydiluted 10-fold in 0.01M sodium phosphate with 0.05% normal human serumat pH 4.8. They were also serially diluted 10-fold in PBS (pH 7.2) with0.01% Tween 20®.

Because it is known that, in general, acridinium compounds are morestable at an acid pH, it was assumed that the counts obtained from thesamples diluted in pH 4.8 buffer would be representative of the maximumstability with maximum chemiluminescent output. All three compounds wereserially diluted 10-fold to a final concentration of 1.6×10⁻¹⁰ M. A 10μl aliquot of each sample was added to 90 μl of 0.05N HCl.Chemiluminescence was triggered with 200 μl of 0.03% H₂ O₂ in 0.25N NaOHand counts were monitored on a luminometer for 6 seconds with results aspresented in Table 3. Results are presented in Table 3 for each of threeruns.

                  TABLE 4                                                         ______________________________________                                        Compound No. Identifying No.                                                                           Counts/6 Seconds                                     ______________________________________                                        62           13513-211   92,669                                                                        91,241                                                                        91,995                                               83           30253-020   138,791                                                                       141,962                                                                       145,133                                              87           13514-020   59,438                                                                        59,443                                                                        59,449                                               ______________________________________                                    

Within experimental error, chemiluminescent output on the luminometerdid not differ among the compounds, as indicated in Table 4.

                  TABLE 5                                                         ______________________________________                                        Chemiluminescent Output at pH 4.8                                             Compound No.  Identifying No.                                                                           Counts/Mole                                         ______________________________________                                        62            13513-211   5.7 × 10.sup.19                               83            30253-020   8.7 × 10.sup.19                               87            13514-020   3.7 × 10.sup.19                               ______________________________________                                    

When 10 μl of these same compounds were diluted to 1.6×10⁻¹⁰ M in 90 μlPBS buffer (pH 7.2) with 0.01% Tween 20® and not acidified prior torunning chemiluminescence output determinations as above, the resultswere somewhat different, especially for the acridinium carboxylatecompound 13514-020, as shown in Table 5. Results are presented in Table5 for each of three runs.

                  TABLE 6                                                         ______________________________________                                        Chemiluminescent Output at pH 7.2                                             Compound No. Identifying No.                                                                           Counts/6 Seconds                                     ______________________________________                                        62           13513-211    88,633                                                                        89,135                                                                        90,394                                              83           30253-020   133,560                                                                       137,929                                                                       142,299                                              87           13514-020    8,185                                                                         7,274                                                                         6,363                                               ______________________________________                                    

The compound identified by the number 13514-020 produced only 4.4×10¹⁸counts/mole in pH 7.2 buffer, almost an order of magnitude fewer countsthan it produced at pH 4.8. This may be due to pseudobase formation by alarge proportion of the molecules at the more alkaline pH, thepseudobase being substantially less chemiluminescent than thecorresponding positively charged acridinium compound.

The N-sulfonylacridinium carboxamide compounds showed only a very smalldrop in counts when incubated at pH 7.2. This suggests that they do notundergo pseudobase formation to any appreciable degree, at least at thispH.

The dilution series of all three of the acridinium compounds in pH 7.2buffer were stored overnight at room temperature and then assayed. BothN-sulfonylacridium carboxamide compounds showed virtually no change inchemiluminescence. The phenyl acridinium carboxylate showed asignificant drop after 20 hours at room temperature.

The samples were then placed in an incubator at 45° C. Every day for theduration of the study they were removed from the incubator, cooled toroom temperature, and 10 μl aliquots diluted into 90 μl of PBS buffer(pH 7.2) were assayed for chemiluminescence.

Neither of the N-sulfonylacridinium carboxamides showed any significantdifference in chemiluminescent output when diluted either in 0.05N HClor in PBS at pH 7.2. However, the acridinium carboxylate 13514-020exhibited a significantly different chemiluminescent output when dilutedin 0.05N HCl or in PBS buffer at pH 7.2. When diluted in PBS buffer (pH7.2), the acridinium carboxylate consistently produced at least 10-foldfewer counts than when diluted in 0.05N HCl.

The 10,N-bis-(3-sulfopropyl) acridinium carboxamide (compound 83,30253-020) appears to be quite stable at pH 7.2 at 45° C. After 10 daysunder such conditions no appreciable loss of chemiluminescence wasobserved. Compound 13513-211 produced 10-fold fewer counts, and theacridinium carboxylate 13514-020 produced 10³ fewer counts under thesame conditions.

EXAMPLE 14 Preparation of Labeled IgG

Disulfopropyl compound 83, 30253-020, was activated by treatment withphosphorous oxychloride in acetonitrile at 45° C. for 12 hours underargon. The solvent and excess POCl₃ were removed in vacuo and theactivated compound was used directly in the labeling reaction.

Thus, 10 mg of rabbit IgG (Sigma Chemical Company, St. Louis, Mo.) wasdissolved in 0.1M sodium phosphate buffer (2 ml, pH 7.0) containing 1%Tween 80® (polyoxyethylenesorbitan momooleate, available from SigmaChemical Company, St. Louis, Mo.). One ml of this solution was mixedwith about 2 mg of the bis-sulfonylchloride. The solution was agitatedperiodically by sonication and stirring for one hour at roomtemperature.

An aliquot (0.5 ml) of the reaction solution was chromotographed overSephadex® G-25 (beads for gel filtration, prepared by cross-linkingdextran with epichlorohydrin, 10 cm×0.75 cm), as available fromPharmacia, Piscataway, N.J., and eluted with 0.1M phosphate buffer (pH6.5).

The labeled protein eluted as a weakly green fluorescent band. Thelabeled protein was further purified by HPLC using a Bio-Sil® TSK-250column (size exclusion column, hydrophilic bonded phase silica BioRad,Richmond, Calif.). The resulting conjugate (30253-34) contained 0.8labels/protein, as determined from the ratio of the absorbance at 370 nm(ε≅10,000, acridinium salt) to the absorbance 280 nm (ε≅210,000, IgG).

EXAMPLE 15 Heat Stability Studies

The conjugate 30253-34, as synthesized in Example 14, was seriallydiluted 10-fold in three buffers (0.1M sodium phosphate, 0.01% Tween20®, pH 6.3; 0.01M sodium phosphate, 0.15M NaCl, 0.01% Tween 20®, pH6.8; and 0.01M sodium phosphate, 0.15M NaCl, 0.01% Tween 20®, pH 7.2) toa concentration of 2×10⁻⁹ M IgG and 1.6×10⁻⁹ M acridinium. A dilutionseries was prepared and initial counts were recorded by taking 10 μl ofthe sample, diluting with 90 μl of PBS buffer at pH 6.3, pH 6.8, or pH7.2, and then triggering chemiluminescence with 200 μl of 0.03% H₂ O₂ in0.25N NaOH. A 100 μl sample of PBS buffer was used as a control for eachseries.

Counts shown in Table 6 are averages of results for duplicate samplesassayed on the day on which the dilution series was prepared. Theconcentration shown in Table 5 is the concentration of the sample priorto dilution. The amount in parentheses for each entry in Table 5 is theamount of conjugate present in the sample.

                  TABLE 7                                                         ______________________________________                                        Concentration (Amount)                                                                              Counts/6 Seconds                                        ______________________________________                                        pH 6.3                                                                        buffer (0 moles)      253                                                     2 × 10.sup.-10 M (2 × 10.sup.-14 moles)                                                 216,054                                                 1 × 10.sup.-10 M (1 × 10.sup.-14 moles)                                                 100,842                                                 5 × 10.sup.-11 M (5 × 10.sup.-15 moles)                                                 48,704                                                  2.5 × 10.sup.-11 M (2.5 × 10.sup.-15 moles)                                             23,771                                                  1.25 × 10.sup.-11 M (1.25 × 10.sup.-15 moles)                                           11,475                                                  6 × 10.sup.-12 M (6 × 10.sup.-16 moles)                                                 5,866                                                   pH 6.8                                                                        buffer (0 moles)      233                                                     2 × 10.sup.-10 M (2 × 10.sup.-14 moles)                                                 295,608                                                 1 × 10.sup.-10 M (1 × 10.sup.-14 moles)                                                 149,725                                                 5 × 10.sup.-11 M (5 × 10.sup.-15 moles)                                                 76,820                                                  2.5 × 10.sup.-11 M (2.5 × 10.sup.-15 moles)                                             38,801                                                  1.25 × 10.sup.-11 M (1.25 × 10.sup.-15 moles)                                           18,408                                                  6 × 10.sup.-12 M (6 × 10.sup.-16 moles)                                                 9,398                                                   pH 7.2                                                                        buffer (0 moles)      726                                                     2 × 10.sup.-10 M (2 × 10.sup.-14 moles)                                                 309,445                                                 1 × 10.sup.-10 M (1 × 10.sup.-14 moles)                                                 156,311                                                 5 × 10.sup.-11 M (5 × 10.sup.-15 moles)                                                 77,238                                                  2.5 × 10.sup.-11 M (2.5 × 10.sup.-15 moles)                                             39,879                                                  1.25 × 10.sup.-11 M (1.25 × 10.sup.-15 moles)                                           19,925                                                  6 × 10.sup.-12 M (6 × 10.sup.-16 moles)                                                 10,526                                                  ______________________________________                                    

Each dilution series was placed in a warm air incubator at 45° C. afteran initial reading was taken. A duplicate reading was made on eachsample daily and then the readings were averaged.

When the conjugate was stored at pH 6.8 and at 45° C., there was no lossin chemiluminescent activity of the label over a 15 day period ofobservation, at any dilution. Essentially the same results were observedwhen the conjugate was stored in PBS buffer at pH 7.2.

EXAMPLE 16 Comparison of CLIA vs. RIA A. Preparation ofAcridinium-Labeled Anti-HsAg Conjugate.

Compound 75 (13514-081, Example 6) (12.5 μmol) was dissolved in 200 μlof DMF, was treated with NHS (dissolved in 50 μl of DMF) anddicyclohexylcarbodiimide (dissolved in 50 μl of DMF) ("DCC"); andstirred for 12 hours at room temperature. The solution of the activatedester was mixed with mouse monoclonal anti-HBsAg in 0.1M sodiumphosphate buffer (pH 6.3) in a molar ratio of 100:1 at 4° C. for 12hours.

The conjugate was then dialysed against PBS buffer, pH 6.3, until theabsorbance of the dialysate indicated no free label. A UV spectralanalysis indicated between 2 to 6 labels/antibody (as determined from aratio of absorbances as in Example 14).

B. Assay for HBsAg.

Either type A_(d) or type A_(y) HBsAg (200 μl) was diluted in calf serumand was reacted with an Auszyme™ (Abbott Laboratories, Abbott Park,Ill.) monoclonal antibody bead and 2×10⁵ of counts of ¹²⁵ I-labeledmouse monoclonal anti-HBsAg antibody (40 μl, in the RIA) or anacridinium-labeled mouse monoclonal anti-HBsAg antibody (40 μl, in theCLIA) in PBS containing 50% calf serum, 10% human serum, 0.05% Tween 20®and 5 mM EDTA (pH 6.3), for three hours at 40° C. The beads were thenwashed 6 times in water and counted for their activities. Calf serum wasused as a negative control.

In the CLIA, a polystyrene bead with conjugate bound adsorbed theretowas mixed with 250 μl phosphate, 0.5 mM, pH 5.3, in a glass vialsuitable for use in a luminometer. While the sample was in the measuringposition, 0.2 ml of 0.03% H₂ O₂ in 0.25N NaOH was then injected into theglass vial. The light emmitted was measured in the luminometer. Readingbegan 0.012 seconds before initiation of the chemical reaction andcontinued for 6 seconds.

The results are presented in Table 7.

                  TABLE 8                                                         ______________________________________                                        Concentration CLIA              RIA                                           (ng/ml)       A.sub.d A.sub.y   A.sub.d                                                                            A.sub.y                                  ______________________________________                                        1.0           2214    3144      371  400                                      0.5           1256    2494      236  408                                      0.25          701     921       221  248                                      0.125         521     592       173  179                                      Calf Serum    151     179                                                     Cut-off       327     376                                                     ______________________________________                                    

Under the stated conditions, the sensitivity for the CLIA was less than0.125 ng/ml for both the A_(d) and A_(y) types of HBsAg. For the RIA thesensitivity was 1.0 ng/ml for both the A_(d) and A_(y) types. Thecut-off count was 2.1 times that of the negative control.

Table 8 clearly shows that chemiluminescent immunoassays according tothe present invention are more sensitive than comparableradioimmunoassays.

EXAMPLE 17 A comparison of CLIA and EIA A. Preparation of labeledanti-hTSH (30234-207).

Compound 75 (13514-081, Example 6) (2 mg, 4.3 μmoles) in 200 ml ofacetonitrile was treated with1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (Sigma, St.Louis, Mo.) (10 μmoles) in 100 μl of acetonitrile andN-hydroxysuccinimide (4-9 μmoles) in 100 μl of acetonitrile for 12 hoursat 25° C. in the dark.

The active ester was mixed with anti-hTSH in PBS buffer containing 0.5%3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate ("CHAPS") atpH 6.5 in a ratio of 50:1 (antibody:active ester). After coupling for 3hours at 25° C., the labeled antibody was dialysed against PBS buffercontaining 0.5% CHAPS at pH 6.5 until no free label was present in thedialysate by U.V.

Based on the U.V. spectra, the conjugate had an average of 10 labels perantibody.

B. Assay for hTSH.

CLIA and EIA were compared Using the Abbott hTSH-EIA Kit (AbbottLaboratories, Abbott Park, Ill.) with the exception that for the CLIA,the anti-hTSH acridininium conjugate was used in place of the kitanti-hTSH-HRPO conjugate. Thus, a standard curve was generated byincubating the kit standards with the kit beads at 37° C. for 1 hour,then washing three times. For the CLIA, the conjugate prepared above wasdiluted 1:5000 with PBS buffer containing 50% calf serum, 1% normalmouse serum, 0.05% Tween® 20 and 2 mM EDTA at pH 6.3. One hundredmicroliters of this solution was incubated with the beads for 1 hour at37° C., then washed four times.

The beads were transferred one by one to the reaction vial of aluminometer containing 400 μl of water and reacted with 200 μl of 0.03%H₂ O₂ in 0.2N NaOH. Photon counts were recorded for 6 seconds.

The EIA was carried out according to the instructions in the kit inserton a Quantum II® spectro photometer (Abbott Laboratories, Abbott Park,Ill.)

The results are shown in Table 9.

                  TABLE 9                                                         ______________________________________                                        Concentration   CLIA       EIA                                                (μIu/ml)     (counts)   (A.sub.492)                                        ______________________________________                                         0               533       0.012                                                              (SD35.4)                                                       1               5064      0.062                                               4              14476      0.176                                              10              32092      0.397                                              25              66072      0.828                                              60              110,984    1.602                                              ______________________________________                                    

Under these conditions the sensitivity of the CLIA was 0.016 μIU/ml (0standard+2 SD) while the EIA had a sensitivity of 0.05 μIU/ml.

EXAMPLE 18 Preparation of5-methyl-6-[N-tosyl-N-(2-carboxyethyl)]-phenanthridiniumcarboxamide

Phenanthridine-6-carboxylic acid (400 mg, 1.8 mmoles) [prepared by themethod of Wittig et al., Justus Liebig's Ann., 577, 1 (1952)], wassuspended in methylene chloride (20 ml, distilled from P₂ O₅) and cooledto 0° C. under nitrogen. Oxalyl chloride (320 μl, 3.6 mmoles) (AldrichChemical Co., Milwaukee, Wis.) was added, followed by DMF (5 μl). As thereaction mixture was stirred for one hour at 0° C. and for 30 minutes at25° C., all the carboxylic acid dissolved. The solution was evaporatedto dryness to give the acid chloride which was used without furtherpurification.

Methyl N-tosyl-β-alanine was prepared from methyl-β-alanine (AldrichChemical Company, Milwaukee, Wis.) and tosyl chloride (Aldrich ChemicalCompany, Milwaukee, Wis.) according to the procedure of Example 1.Potassium t-butoxide (600 mg, 5.4 mmoles, freshly sublimed) was added toa solution of 1.3g (5.4 mmoles) of methyl N-tosyl-β-alanine in 50 ml ofTHF. After stirring for 15 minutes and at room temperature and under N₂,the suspension was evaporated to dryness. The potassium salt of methylN-tosyl-β-alanine, was resuspended in 20 ml of THF, mixed with the acidchloride (in 20 ml of THF), and stirred for 12 hours.

The resulting suspension was poured into 100 ml of ethyl acetate, washedwith 50 ml of water and washed twice with 25 ml of brine. After dryingover MgSO₄ and evaporating to dryness, the residue was chromatographedon a Chromatatron™ chromatograph (available from Harrison Research, PaloAlto, Calif.) using a 4 mm silica rotor and employing a 25/75ethylacetate/hexane gradient. The product (R_(f) 0.2) was collected,then recrystallized from benzene/hexane (i.e., the product was dissolvedin benzene, and hexane was added until cloudy) to give 130 mg of methyl6-[N-tosyl-N-(2-carboxyethyl)]phenanthridinecarboxamide, Compound 84,13514-225.

Compound 84, 13514-225, was methylated according to the procedure inExample 3 to give methyl5-methyl-6-[N-tosyl-N-(2-carboxyethyl)]phenanthridiniumcarboxamide,compound 85, 13514-227. Compound 85 was hydrolyzed according to theprocedure in Example 8 to provide5-methyl-6-[N-tosyl-N-(2-carboxyethyl)]-phenanthridiniumcarboxamide,compound 86, 13514-228.

Compounds 84, 85 and 86 are described using the numerals, symbols andabbreviations as explained in Example 1.

    ______________________________________                                        84.    13514-225   Methyl 6-[N-tosyl-                                                            N-(2-carboxyethyl)]-                                                          phenanthridinecarboxylate                                                     MS M + H @ 463                                             85.    13514-227   Methyl 5-methyl-                                                              6-[N-tosyl-N-                                                                 (2-carboxyethyl)]-                                                            phenanthridiniumcarboxamide                                                   MS M.sup.+ @ 477                                                              M.sub.p 136° C.                                     86.    13514-228   5-Methyl-6-[N-tosyl-                                                          N-(2-carboxyethyl)]-                                                          phenanthridiniumcarboxamide                                                   MS M.sup.+ @ 463                                           ______________________________________                                    

Although the present invention has been described in terms of preferredembodiments, it is understood that modifications and improvements willoccur to those skilled in the art.

For example, in light of the results presented herein, it is expectedthat additional compounds which are useful according to the presentinvention may be identified by the formula ##STR4##

wherein a, b, c, d, a¹, b¹, c¹, d¹ independently may be hydrogen, alkyl,aryl, amino, substituted amino, carboxy-alkyl, sulfoalkyl, alkoxyl,aryloxy, sulfo, thio alkoxyl, thioaryloxy aminoalkyl, protectedaminoalkyl, hydroxyalkyl, protected hydroxyalkyl, haloalkyl, or anyadjacent of these positions may be linked so as to form aromatic ringsfused to the acridine nucleus.

In addition, Sheehan et al., U.S. Pat. No. 3,539,574 describeschemiluminescent acridinium compounds which are also expected to beuseful according to the present invention. Other isomericacridinecarboxylic acids, quinoline carboxylic acids, isoquinolinecarboxylic acid, other activated acridine amides, and other activatedacridine esters are expected to be useful according to the presentinvention. Such compounds include, without limitation: hydroxamatesidentified by the formula ##STR5## enamides identified by the formula##STR6## arylamides identified by the formula ##STR7## wherein X and Y¹are electron withdrawing groups; N-heterocycles identified by theformula ##STR8## wherein X and Y¹ may independently be O, S, P, N, or C;activated esters such as thiolesters identified by the formula ##STR9##or such as thioesters identified by the formula ##STR10## acridine acidsidentified by the formula ##STR11## or by the formula ##STR12##quinoline acids identified by the formula ##STR13## or by the formula##STR14## or isoquinoline acids identified by the formula ##STR15## orby the formula ##STR16##

It is understood that those skilled in the art will be enabled by theabove specification to incorporate reactive functional groups forattaching the label to an analyte into compounds according to thepresent invention.

It is also contemplated that compounds according to the presentinvention will be: used in labeling DNA probes; incorporated into anenzyme substrate wherein the product of the enzymatic reaction is thechemiluminescent compound; and incorporated into systems which involveenergy transfer or fluorescent quenching.

Compounds according to the present invention may also be: incorporatedinto a system which employs the compound as a labeling reagent in apost-column HPLC detection system; used to measure H₂ O₂ in otheranalytical systems, including the quantitation of intracellular H₂ O₂concentration; and used as a source of intense pulsed light.

Therefore, it is intended that the present invention include all suchvariations and improvements as come within the scope of the invention asclaimed.

What is claimed is:
 1. A chemiluminescent conjugate comprising anantibody conjugated to a chemiluminescent compound of the formula:##STR17## wherein R, R', R", X¹, X², and X³ independently comprisesubstituents which do not interfere with chemiluminescence measurement,linkage to the antibody is through one of X¹, X² and X³, and wherein Y--is an anion with the proviso that R-X³, R'-X² and R"-X¹ may behydrogen,wherein X¹, X² and X³ are independently members of the groupconsisting of hydrogen, (--CO₂ H), (--C(═O)OZ¹) wherein Z¹ is alkyl,(--C(═O)NZ² Z³) wherein Z² or Z³ is H, alkyl or aryl, (--C(═O)OZ⁴)wherein Z⁴ is aryl, (--CN), (--C(═O)N(Z⁵)--C(═O)--Z⁵) wherein Z⁵ is H,alkyl or aryl, (F, Cl, Br, or I), (--N═C═O), (--N═C═S), (--SO₃ ⁻),sulfonyl halide (--SO₂ Z⁶) wherein Z⁶ is (Cl or F), (--C(═O)--Z⁷)wherein Z⁷ is (F, Cl, or Br), ##STR18## groups; wherein R independentlycomprises a member selected from the group consisting of alkylene,alkenylene and substituted alkylene groups and wherein R' and R"independently comprise a member selected from the group consisting ofalkylene, arylene, alkenylene, substituted alkylene, and substitutedarylene groups, such that: one or more hydrogens or open valences ofsaid member is replaced by an alkyl, aryl, alkylene, alkenylene,substituted alkyl, substituted alkylene, substituted aryl, alkoxy,aryloxy, halo, nitro, amino, protected amino, substituted amino,hydroxy, protected hydroxy, oxo, thio, imino, mercapto or substitutedmercapto group; or such that one or more carbon atoms of the member isreplaced by a heteroatom.
 2. In a method for performing achemiluminescent assay to test for the presence of a member of aspecific binding pair which may be present in a test sample wherein saidmethod comprises (a) contacting said test sample suspected of containingsaid specific binding pair member with a conjugate comprising anantibody attached to a chemiluminescent compound capable of generating adetectable signal, which conjugate specifically reacts with saidspecific binding pair member and (b) determining the presence of saidspecific binding pair member by detecting the signal generated by saidconjugate, wherein the improvement comprises the step of contacting saidtest sample suspected of containing said specific binding pair memberwith said chemiluminescent conjugate as recited in claim 92 wherein saidantibody of said conjugate specifically binds said specific binding pairmember.
 3. A chemiluminescent conjugate comprising an antigen conjugatedto a chemiluminescent compound of the formula: ##STR19## wherein R, R',R", X¹, X², and X³ independently comprise substituents which do notinterfere with chemiluminescence measurement, linkage to the antigen isthrough one of X¹, X² and X³, and wherein Y-- is an anion with theproviso that R-X³, R'-X² and R"-X¹ may be hydrogen,wherein X¹, X² and X³are independently members of the group consisting of hydrogen, (--CO₂H), (--C(═O)OZ¹) wherein Z¹ is alkyl, (--C(═O)NZ² Z³) wherein Z² or Z³is H, alkyl or aryl, (--C(═O)OZ⁴) wherein Z⁴ is aryl, (--CN),(--C(═O)N(Z⁵)--C(═O)--Z⁵) wherein Z⁵ is H, alkyl or aryl, (F, Cl Br, orI), (--N═C═O), (--N═C═S), (--SO₃ ⁻), sulfonyl halide (--SO₂ Z⁶) whereinZ⁶ is (Cl or F), (--C(═O)--Z⁷) wherein Z⁷ is (F, Cl, or Br), ##STR20##groups; wherein R independently comprises a member selected from thegroup consisting of alkylene, alkenylene and substituted alkylene groupsand wherein R' and R" independently comprise a member selected from thegroup consisting of alkylene, arylene, alkenylene, substituted alkylene,and substituted arylene groups, such that: one or more hydrogens or openvalences of said member is replaced by an alkyl, aryl, alkylene,alkenylene, substituted alkyl, substituted alkylene, substituted aryl,alkoxy, aryloxy, halo, nitro, amino, protected amino, substituted amino,hydroxy, protected hydroxy, oxo, thio, imino, mercapto or substitutedmercapto group; or such that one or more carbon atoms of the member isreplaced by a heteroatom.
 4. In a method for performing achemiluminescent assay to test for the presence of a member of aspecific binding pair which may be present in a test sample wherein saidmethod comprises (a) contacting said test sample suspected of containingsaid specific binding pair member with a conjugate comprising an antigenattached to a chemiluminescent compound capable of generating adetectable signal, which conjugate specifically reacts with saidspecific binding pair member and (b) determining the presence of saidspecific binding pair member by detecting the signal generated by saidconjugate, wherein the improvement comprises the step of contacting saidtest sample suspected of containing said specific binding pair memberwith said chemiluminescent conjugate as recited in claim 3 wherein saidantigen of said conjugate specifically binds said specific binding pairmember.
 5. A chemiluminescent conjugate comprising a polynucleotideconjugated to a chemiluminescent compound of the formula: ##STR21##wherein R, R', R", X¹, X², and X³ independently comprise substituentswhich do not interfere with chemiluminescence measurement, linkage tothe polynucleotide is through one of X¹, X² and X³, and wherein Y-- isan anion with the proviso that R-X³, R'-X² and R"-X¹ may behydrogen,wherein X¹, X² and X³ are independently members of the groupconsisting of hydrogen, (--CO₂ H), (--C(═O)OZ¹) wherein Z¹ is alkyl,(--C(═O)NZ² Z³) wherein Z² or Z³ is H, alkyl or aryl, (--C(═O)OZ⁴)wherein Z⁴ is aryl, (--CN), (--C(═O)N(Z⁵)--C(═O)--Z⁵) wherein Z⁵ is H,alkyl or aryl, (F, Cl Br, or I), (--N═C═O), (--N═C═S), (--SO₃ ⁻),sulfonyl halide (--SO₂ Z⁶) wherein Z⁶ is (Cl or F), (--C(═O)--Z⁷)wherein Z⁷ is (F, Cl, or Br), ##STR22## groups; wherein R independentlycomprises a member selected from the group consisting of alkylene,alkenylene and substituted alkylene groups and wherein R' and R"independently comprise a member selected from the group consisting ofalkylene, arylene, alkenylene, substituted alkylene, and substitutedarylene groups, such that: one or more hydrogens or open valences ofsaid member is replaced by an alkyl, aryl, alkylene, alkenylene,substituted alkyl, substituted alkylene, substituted aryl, alkoxy,aryloxy, halo, nitro, amino, protected amino, substituted amino,hydroxy, protected hydroxy, oxo, thio, imino, mercapto or substitutedmercapto group; or such that one or more carbon atoms of the member isreplaced by a heteroatom.
 6. In a method for performing achemiluminescent assay to test for the presence of a polynucleotidewhich may be present in a test sample wherein said method comprises (a)contacting said test sample suspected of containing said polynucleotidewith a conjugate comprising a polynucleotide complementary to saidpolynucleotide in said test sample which complementary polynucleotidespecifically reacts with said polynucleotide of said test sample andwhich complementary polynucleotide is attached to a chemiluminescentcompound capable of generating a detectable signal and (b) determiningthe presence of said polynucleotide by detecting the signal generated bysaid conjugate, wherein the improvement comprises the step of contactingsaid test sample suspected of containing said polynucleotide with saidchemiluminescent conjugate as recited in claim 96 which polynucleotideof said conjugate is complementary to said polynucleotide in said testsample and wherein said complementary polynucleotide of said conjugatehybridizes with said polynucleotide in said test sample.
 7. Achemiluminescent conjugate comprising a non-antibody, binding-proteinconjugated to a chemiluminescent compound of the formula: ##STR23##wherein R, R', R", X¹, X², and X³ independently comprise substituentswhich do not interfere with chemiluminescence measurement, linkage tothe non-antibody, binding-protein is through one of X¹, X² and X³, andwherein Y-- is an anion with the proviso that R-X³, R'-X² and R"-X¹ maybe hydrogen,wherein X¹, X² and X³ are independently members of the groupconsisting of hydrogen, (--CO₂ H), (--C(═O)OZ¹) wherein Z¹ is alkyl,(--C(═O)NZ² Z³) wherein Z² or Z³ is H, alkyl or aryl, (--C(═O)OZ⁴)wherein Z⁴ is aryl, (--CN), (--C(═O)N(Z⁵)--C(═O)--Z⁵) wherein Z⁵ is H,alkyl or aryl, (F, Cl Br, or I), (--N═C═O), (--N═C═S), (--SO₃ ⁻),sulfonyl halide (--SO₂ Z⁶) wherein Z⁶ is (Cl or F), (--C(═O)--Z⁷)wherein Z⁷ is (F, Cl, or Br), ##STR24## groups; wherein R independentlycomprises a member selected from the group consisting of alkylene,alkenylene and substituted alkylene groups and wherein R' and R"independently comprise a member selected from the group consisting ofalkylene, arylene, alkenylene, substituted alkylene, and substitutedarylene groups, such that: one or more hydrogens or open valences ofsaid member is replaced by an alkyl, aryl, alkylene, alkenylene,substituted alkyl, substituted alkylene, substituted aryl, alkoxy,aryloxy, halo, nitro, amino, protected amino, substituted amino,hydroxy, protected hydroxy, oxo, thio, imino, mercapto or substitutedmercapto group; or such that one or more carbon atoms of the member isreplaced by a heteroatom.
 8. In a method for performing achemiluminescent assay to test for the presence of a complementaryanalyte of a non-antibody, binding-protein which may be present in atest sample wherein said method comprises (a) contacting said testsample suspected of containing said complementary analyte of saidnon-antibody, binding protein with a conjugate comprising anon-antibody, binding protein which specifically reacts with saidcomplementary analyte of said non-antibody, binding protein and whichnon-antibody, binding-protein is attached to a chemiluminescent compoundcapable of generating a detectable signal and (b) determining thepresence of said complementary analyte of said non-antibody, bindingprotein by detecting the signal generated by said conjugate, wherein theimprovement comprises the step of contacting said test sample suspectedof containing said complementary analyte with said chemiluminescentconjugate as recited in claim 7 wherein said non-antibody,binding-protein of said conjugate specifically binds said complementaryanalyte.
 9. A chemiluminescent conjugate comprising a hapten conjugatedto a chemiluminescent compound of the formula: ##STR25## wherein R, R',R", X¹, X², and X³ independently comprise substituents which do notinterfere with chemiluminescence measurement, linkage to the hapten isthrough one of X¹, X² and X³, and wherein Y-- is an anion with theproviso that R-X³, R'-X² and R"-X¹ may be hydrogen,wherein X¹, X² and X³are independently members of the group consisting of hydrogen, (--CO₂H), (--C(═O)OZ¹) wherein Z¹ is alkyl, (--C(═O)NZ² Z³) wherein Z² or Z³is H, alkyl or aryl, (--C(═O)OZ⁴) wherein Z⁴ is aryl, (--CN),(--C(--O)N(Z⁵)--C(═O)--Z⁵) wherein Z⁵ is H, alkyl or aryl, (F, Cl, Br,or I), (--N═C═O), (--N--C═S), (--SO₃ ⁻), sulfonyl halide (--SO₂ Z⁶)wherein Z⁶ is (Cl or F), (--C(═O)--Z⁷) wherein Z⁷ is (F, Cl, or Br),##STR26## groups; wherein R independently comprises a member selectedfrom the group consisting of alkylene, alkenylene and substitutedalkylene groups and wherein R' and R" independently comprise a memberselected from the group consisting of alkylene, arylene, alkenylene,substituted alkylene, and substituted arylene groups, such that: one ormore hydrogens or open valences of said member is replaced by an alkyl,aryl, alkylene, alkenylene, substituted alkyl, substituted alkylene,substituted aryl, alkoxy, aryloxy, halo, nitro, amino, protected amino,substituted amino, hydroxy, protected hydroxy, oxo, thio, imino,mercapto or substituted mercapto group; or such that one or more carbonatoms of the member is replaced by a heteroatom.
 10. In a method forperforming a competitive chemiluminescent assay to test for the presenceof a hapten which may be present in a test sample wherein said methodcomprises (a) contacting said test sample suspected of containing saidhapten with a known amount of an anti-hapten antibody specific for saidhapten and with a conjugate comprising a hapten which competes with saidhapten in said test sample for binding with said anti-hapten antibodyand wherein said hapten of said conjugate is attached to achemiluminescent compound capable of generating a detectable signal and(b) determining the presence of said hapten in said test sample bydetecting the signal generated by said conjugate, wherein theimprovement comprises the step of contacting said test sample suspectedof containing said hapten with said chemiluminescent conjugate asrecited in claim 9 wherein said hapten of said conjugate competes withsaid hapten in said test sample for specific binding to said anti-haptenantibody.
 11. A chemiluminescent conjugate comprising a small molecularweight analyte which binds to a binding protein conjugated to achemiluminescent compound of the formula: ##STR27## wherein R, R', R",X¹, X², and X³ independently comprise substituents which do notinterfere with chemiluminescence measurement, linkage to the smallmolecular weight analyte is through one of X¹, X² and X³, and whereinY-- is an anion with the proviso that R-X³, R'-X² and R"-X¹ may behydrogen,wherein X¹, X² and X³ are independently members of the groupconsisting of hydrogen, (--CO₂ H), (--C(═O)OZ¹) wherein Z¹ is alkyl,(--C(═O)NZ² Z³) wherein Z² or Z³ is H, alkyl or aryl, (--C(═O)OZ⁴)wherein Z4 is aryl, (--CN), (--C(═O)N(Z⁵)--C(--O)--Z⁵) wherein Z⁵ is H,alkyl or aryl, (F, Cl, Br, or I), (--N═C═O), (--N═C═S), (--SO₃ ⁻),sulfonyl halide (--SO₂ Z⁶) wherein Z⁶ is (Cl or F), (--C(═O)--Z⁷)wherein Z⁷ is (F, Cl, or Br), ##STR28## groups; wherein R independentlycomprises a member selected from the group consisting of alkylene,alkenylene and substituted alkylene groups and wherein R' and R"independently comprise a member selected from the group consisting ofalkylene, arylene, alkenylene, substituted alkylene, and substitutedarylene groups, such that: one or more hydrogens or open valences ofsaid member is replaced by an alkyl, aryl, alkylene, alkenylene,substituted alkyl, substituted alkylene, substituted aryl, alkoxy,aryloxy, halo, nitro, amino, protected amino, substituted amino,hydroxy, protected hydroxy, oxo, thio, imino, mercapto or substitutedmercapto group; or such that one or more carbon atoms of the member isreplaced by a heteroatom.
 12. In a method for performing a competitivechemiluminescent assay to test for the presence of a small molecularweight analyte which binds to a binding protein and which may be presentin a test sample wherein said method comprises (a) contacting said testsample suspected of containing said small molecular weight analyte witha known amount of said binding protein specific for said small molecularweight analyte and with a conjugate comprising a small molecular weightanalyte which competes with said small molecular weight analyte in saidtest sample and which binds to said binding protein, wherein said smallmolecular weight analyte of said conjugate is attached to achemiluminescent compound capable of generating a detectable signal and(b) determining the presence of said small molecular weight analyte bydetecting the signal generated by said conjugate, wherein theimprovement comprises the step of contacting said test sample suspectedof containing said small molecular weight analyte with saidchemiluminescent conjugate as recited in claim 11 wherein said smallmolecular weight analyte of said conjugate competes with said smallmolecular weight analyte in said test sample for specific binding tosaid binding protein.
 13. A chemiluminescent conjugate formed by amember selected from the group consisting of an antigen, an antibody, ahapten, a polynucleotide, a small molecular weight analyte which bindsto a binding protein and a non-antibody, binding-protein conjugated to achemiluminescent compound of the formula: ##STR29## wherein Rindependently comprises a member selected from the group consisting ofalkylene, alkenylene and substituted alkylene groups such that:one ormore hydrogens or open valences of said member is replaced by an alkyl,aryl, alkylene, alkenylene, substituted alkyl, substituted alkylene,substituted aryl, alkoxy, aryloxy, halo, nitro, amino, protected amino,substituted amino, hydroxy, protected hydroxy, oxo, thio, imino,mercapto or substituted mercapto group; or such that one or more carbonatoms of said member is replaced by a heteroatom; Y-- is an anion;wherein conjugation is through X³ and wherein X³ is independently amember of the group consisting of hydrogen, (--CO₂ H), (--C(═O)OZ¹)wherein Z¹ is alkyl, (--C(═O)NZ² Z³) wherein Z² or Z³ is H, alkyl oraryl, (--C(═O)OZ⁴) wherein Z⁴ is aryl, (--CN), (--C(═O)N(Z⁵)--C(═O)--Z⁵)wherein Z⁵ is H, alkyl or aryl, (F, Cl, Br, or I), (--N═C═O), (--N═C═S),(--SO₃ ⁻), sulfonyl halide (--SO₂ Z⁶) wherein Z⁶ is (Cl or F),(--C(═O)--Z⁷) wherein Z⁷ is (F, Cl, or Br), ##STR30## groups; and withthe proviso that R-X³ may also be hydrogen.
 14. The chemiluminescentconjugate as recited in claim 13 wherein said heteroatom is selectedfrom the group consisting of nitrogen, phosphorus, sulfur and oxygen.15. The chemiluminescent conjugate as recited in claim 13 wherein Rindependently is of the formula --(CH₂)_(n) --, where n=0-50.
 16. Amethod for performing a chemiluminescent immunoassay to test for thepresence of an analyte which may be present in a test samplecomprising:(a) contacting said test sample suspected of containing saidanalyte with a chemiluminescent conjugate that specifically binds saidanalyte and which conjugate comprises a member selected from the groupconsisting of an antigen, an antibody, a hapten, a polynucleotide, asmall molecular weight analyte which binds to a binding protein and anon-antibody, binding-protein conjugated through X³ of achemiluminescent compound of the formula: ##STR31## wherein Rindependently comprises a member selected from the group consisting ofalkylene, alkenylene and substituted alkylene groups such that: one ormore hydrogens or open valences of said member is replaced by an alkyl,aryl, alkylene, alkenylene, substituted alkyl, substituted alkylene,substituted awl, alkoxy, aryloxy, halo, nitro, amino, protected amino,substituted amino, hydroxy, protected hydroxy, oxo, thio, imino,mercapto or substituted mercapto group; or such that one or more carbonatoms of said member is replaced by a heteroatom; Y-- is an anion;wherein X³ is independently a member of the group consisting ofhydrogen, (--CO₂ H), (--C(═O)OZ¹) wherein Z¹ is alkyl, (--C(═O)NZ² Z³)wherein Z² or Z³ is H, alkyl or aryl, (--C(═O)OZ⁴) wherein Z⁴ is aryl,(--CN), (--C(═O)N(Z⁵)--C(═O)--Z⁵) wherein Z⁵ is H, alkyl or aryl, (F,Cl, Br, or I), (--N═C═O), (--N═C═S), (--SO₃ ⁻), sulfonyl halide (--SO₂Z⁶) wherein Z⁶ is (Cl or F), (--C(═O)--Z⁷) wherein Z⁷ is (F, Cl, or Br),##STR32## groups; and with the proviso that R-X³ may also be hydrogen;and (b) detecting a signal generated from said chemiluminescentconjugate as an indication of the presence of said analyte in said testsample.
 17. The method as recited in claim 16 wherein said heteroatom ofsaid chemiluminescent conjugate is selected from the group consisting ofnitrogen, phosphorus, sulfur and oxygen.
 18. The method as recited inclaim 13 wherein R of said chemiluminescent conjugate independently isof the formula --(CH₂)_(n) --, where n=0-50.
 19. A chemiluminescentconjugate comprising a member selected from the group consisting of anantigen, an antibody, a hapten, a polynucleotide, a small molecularweight analyte which binds to a binding protein and a non-antibody,binding-protein conjugated through either the CO₂ H or SO₃ ⁻ group of achemiluminescent compound of the formula: ##STR33## wherein Y-- is ananion.
 20. A method for performing a chemiluminescent immunoassay totest for the presence of an analyte which may be present in a testsample comprising:(a) contacting said test sample suspected ofcontaining said analyte with a chemiluminescent conjugate thatspecifically binds said analyte and which conjugate comprises a memberselected from the group consisting of an antigen, an antibody, a hapten,a polynucleotide, a small molecular weight analyte which binds to abinding protein and a non-antibody, binding-protein conjugated througheither the CO₂ H or SO₃ ⁻ group of a chemiluminescent compound of theformula: ##STR34## wherein Y-- is an anion; and (b) detecting a signalgenerated from said chemiluminescent conjugate as an indication of thepresence of said analyte in said test sample.
 21. A chemiluminescentconjugate comprising a member selected from the group consisting of anantigen, an antibody, a hapten, a polynucleotide, a small molecularweight analyte which binds to a binding protein and a non-antibody,binding-protein conjugated through either SO₃ ⁻ group of achemiluminescent compound of the formula: ##STR35## wherein Y-- is ananion.
 22. A method for performing a chemiluminescent immunoassay totest for the presence of an analyte which may be present in a testsample comprising:(a) contacting said test sample suspected ofcontaining said analyte with a chemiluminescent conjugate thatspecifically binds said analyte and which conjugate comprises a memberselected from the group consisting of an antigen, an antibody, a hapten,a polynucleotide, a small molecular weight analyte which binds to abinding protein and a non-antibody, binding-protein conjugated througheither SO₃ ⁻ group of a chemiluminescent compound of the formula:##STR36## wherein Y-- is an anion; and (b) detecting a signal generatedfrom said chemiluminescent conjugate as an indication of the presence ofsaid analyte in said test sample.
 23. A chemiluminescent conjugatecomprising a member selected from the group consisting of an antigen, anantibody, a hapten, a polynucleotide, a small molecular weight analytewhich binds to a binding protein and a non-antibody, binding-proteinconjugated through the CO₂ H group of a chemiluminescent compound of theformula selected from the group consisting of: ##STR37## wherein Y-- isan anion.
 24. A method for performing a chemiluminescent immunoassay totest for the presence of an analyte which may be present in a testsample comprising:(a) contacting said test sample suspected ofcontaining said analyte with a chemiluminescent conjugate thatspecifically binds said analyte and which conjugate comprises a memberselected from the group consisting of an antigen, an antibody, a hapten,a polynucleotide, a small molecular weight analyte which binds to abinding protein and a non-antibody, binding-protein conjugated throughthe CO₂ H group of a chemiluminescent compound of the formula selectedfrom the group consisting of: ##STR38## wherein Y-- is an anion; and (b)detecting a signal generated from said chemiluminescent conjugate as anindication of the presence of said analyte in said test sample.
 25. Achemiluminescent conjugate comprising a member selected from the groupconsisting of an antigen, an antibody, a hapten, a polynucleotide, asmall molecular weight analyte which binds to a binding protein and anon-antibody, binding-protein conjugated through either the CO₂ H or SO₃-- group of a chemiluminescent compound of the formula selected from thegroup consisting of: ##STR39## wherein Y-- is an anion.
 26. A method forperforming a chemiluminescent immunoassay to test for the presence of ananalyte which may be present in a test sample comprising:(a) contactingsaid test sample suspected of containing said analyte with achemiluminescent conjugate that specifically binds said analyte andwhich conjugate comprises a member selected from the group consisting ofan antigen, an antibody, a hapten, a polynucleotide, a small molecularweight analyte which binds to a binding protein and a non-antibody,binding-protein conjugated through either the CO₂ H or SO₃ -group of achemiluminescent compound of the formula selected from the groupconsisting of: ##STR40## wherein Y-- is an anion; and (b) detecting asignal generated from said chemiluminescent conjugate as an indicationof the presence of said analyte in said test sample.